Screening and characterization of sex-specific sequences through 2b-RAD sequencing in American shad (Alosa sapidissima)

American shad (Alosa sapidissima), introduced from the United States, has become one of the most expensive farmed fish in the aquatic product market of China. The shad reveals significant sexual dimorphism in growth and behaviors. For the study, five male-specific tags were identified in two-generation breeding populations of Alosa sapidissima and were verified by PCR amplification. Averages of 10,245,091 and 8,685,704 raw and enzyme reads were obtained by high-throughput sequencing of the 2b-RAD library, respectively. 301,022 unique tags were obtained from the sequences of twenty samples with sequencing depths of 0 to 500. Finally, 274,324 special tags and 29,327 SNPs were selected with a sequencing depth of 3 to 500. Eleven preliminary screening male-specific tags and three male heterogametic SNP loci were isolated. After verification by PCR amplification, five male-specific sequences of 27 bp located on chromosome 3 were screened out. Chromosome 3 could be assumed to be the sex chromosome of Alosa sapidissima. Sex-specific markers will provide invaluable and systematic animal germplasm resources to allow for the precise identification of neo-males for the all-female breeding of Alosa sapidissima in commercial aquaculture.


Introduction
The growth indicators and time of sexual maturity are significantly different between sexes in fish species [1,2]. Culturing single-gender populations will improve both the commercial value and production of fish in aquaculture [2,3]. Since some farmed fish show sexual dimorphism in many characteristics, such as growth indicators or sexual maturity, understanding the sex determination system of these fish will help the aquaculture industry produce mono-sex fish. Genetic mechanisms of determining sex are diverse in fishes [4]. Male heterogamy (XX/XY) and female heterogamy (ZZ/ZW) are two kinds of sex determination systems in fish. The sex determination systems of XX/XO and ZO/ZZ have been found in fish these years [2]. Studying the sex determination systems and selecting sex-specific tags are beneficial for performing mono-sex control breeding [5]. Traditional technologies (amplified fragment length polymorphism (AFLP), simple sequence repeats (SSRs) [6] and random amplified polymorphic DNA a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 fingerprinting (RAPD) [7] have been successful in identifying different sex-specific markers in some aquaculture fishes [8]. However, conventional methods are inefficient and time-consuming. Therefore, next-generation sequencing (NGS) technology has become widespread and provided efficient sex-related markers. Sex-specific DNA fragments and single nucleotide polymorphisms (SNPs) in vertebrates are usually identified by restriction site-associated DNA sequencing (RAD-seq), which is considered an efficient and time-saving technology. The sexspecific markers have played an essential role in the sex identification of fish, such as the zigzag eel (Mastacembelus armatus), redtail catfish (Mystus wyckioides), Ussuri catfish (Pseudobagrus ussuriensis) and Nile tilapia (Oreochromis niloticus) [9][10][11][12][13].
American shad (Alosa sapidissima) is an essential fishery species naturally distributed in North America [14]. The shad were introduced to China in 2003 and successfully cultured in freshwater indoor systems for nearly 20 years in China [15]. The shad has become one of the most expensive farmed species in China, which has prompted an upsurge in consumption [16]. However, shad has quite distinctive dimorphism in body size and behaviors between sexes. American shad males have less commercial value due to both a smaller body size and growth rate than females [15]. Males are trouble makers that have vital chasing behaviors during spring seasons, causing severe body damage to the vulnerable fish and weight loss. Thus, it is crucial to develop an all-female culture population to take advantage of female growth superiority and avoid the risk of injury and weight loss caused by the sexual chasing behavior of males. Therefore, developing sex identification technology for identifying neo-males from sexually reversed mixed males has become a critical measure to develop an all-female population to increase economic output in shad aquaculture.
For the study, 2b-RAD-seq from 60 samples was carried out to show the sexual differences at the molecular level. Finally, five male-specific genotype tags were identified by PCR testing in many farmed mature fish. The research will supplement the data for all-female breeding and sex-specific gene in American shad.

Sample collection
American shad was sampled from Suzhou Fishseeds Biotechnology in Suzhou, China. Ten males and ten females at 20 months old from the broodstock were randomly selected in August 2020 to identify by 2b-RAD sequencing. Twenty males and twenty females of the second generation were randomly sampled for marker validation in June 2021. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The testis, ovaries, and muscle tissue were quickly taken by the surgery after anesthesia. The sex of individuals was determined by sexual organs after anatomy. Muscular tissues were sampled and stored at -20˚C. The total genomic DNA was extracted using Tissue DNA Extraction Kit (Catalog Number DR0301050). Then agarose electrophoresis and UV spectrophotometry (Hangzhou Youmi, Unano-1000) were used to test DNA quality.

2b-RAD library preparation and data analysis
Qingdao OE Biotech Co., Ltd. carried out the analysis of the 2b-RAD library (Qingdao, China), as referred to Wang et al. (2016) [17]. The details of library preparation and data analysis were shown in S1 Text.

Screening of sex-specific sequences
The preliminary screening male-specific tags were 27 bp, which was not long enough for primer design. A chromosomal-level genome assembly of American shad was performed in our previous studies. The genome assembly number described in this paper is JAHTKL01000 0000 in NCBI. According to the location of the tag, the upstream and downstream 300-500 bp sequences of the tag were extracted and the location of the tag was marked. Finally, the primers were designed based on the upstream and downstream 300-500 bp of the tag.

Identification of sex-specific markers
This experiment referred to Zhu et al., 2021 [12]. 14 pairs of primers (Premier 5.0 software) were designed according to eleven sex-specific markers and three sex-specific SNPs (shown in S1 Table) to identify the male-specific 2b-RAD markers. Firstly, PCR amplified was based on fourteen pairs of primers in twenty fish in F0 generation. Then the validated primers were further amplified in forty fish in the second generation. The details of the total PCR volume and amplification program were shown in S2 Text.

Example ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Laboratory Animal Ethics Committee of Suzhou Jianyuan Technology Co., LTD. (Protocol Number: 14393). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

The data of 2b-RAD sequencing
The genome sequences of Alosa sapidissima (version JAHTKL010000000) were chosen as the reference genome. After high-throughput sequencing, averages of 10,245,091 and 8,685,704 raw and enzyme reads were obtained, respectively (shown in Table 1). 301,022 unique tags with sequencing depths of 0 to 500 were obtained. 274,324 tags and 29,327 SNPs were obtained after the removal of unique tags with a sequencing depth of 3 to 500. The distribution of the SNPs on the chromosomes is shown in Fig 1. The proportion of unique tags ranged from 65.07% to 67.41% per sample, and the average sequencing depth was 20.77× (3<depth<500). The result reveals valuable genomics data for identifying sex-specific tags and SNPs.

Identification of sex-specific markers and SNPs
The preliminary screening of eleven male-specific tags ( Table 2) and three heterogametic SNP loci (Table 3) were obtained. The male specificity of the preliminary screening sex-specific markers was verified by a basic local alignment search tool (BLAST) search of the Alosa sapidissima genome. The result showed that the preliminary screening tags and SNP loci revealed

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad 100% match to the reference gene. The male-specific sequences were identified and compared with the assembled genome sequence of male Alosa sapidissima (JAHTKL010000000) obtained from NCBI by BLAST. Then, 14 pairs of primers were designed to test the preliminary screening markers and SNPs (S1 Table). Finally, five male-specific tags were confirmed by PCR in twenty samples. The five male-specific sequences with lengths of 350 bp, 463 bp, 430 bp, 170 bp and 185 bp are shown in Table 4. The male-specific primers of five tags (Tag8736997, Tag8743751, Tag8818409, Tag8821444 and Tag8821742) were shown in Table 5. The results indicated that the male-specific primers of five tags could only amplify bands in male fish (Fig 2).

Verification of male-specific tags in F1 generation
The accuracy of five male-specific tags was identified by the F1 generation. Twenty male and twenty female samples with sex confirmed by sexual organs were obtained for PCR validation. As a result, the male-specific 350 bp, 463 bp, 185 bp, 430 bp and 170 bp sequences were amplified in male fish by the primers Tag8736997 (Fig 3), Tag8743751 (Fig 4), Tag8821742 (Fig 5), Tag8818409 ( Fig 6) and Tag8821444 (Fig 7), respectively. But these verified male-specific tags need further identification in wild Alosa sapidissima. However, this research can supplement genetic information for mono-sex control breeding in Alosa sapidissima.

Discussion
For aquaculture, sex-controlled breeding and monosex culturing are essential to ensure both yield and nutrient requirements. Sex determination systems in fishes are various, with heterogametic males (XY) or heterogametic females (ZW) [18]. Determining the sex determination system in fishes is difficult because of the highly similar size and low differentiation of heteromorphic sex chromosomes [19]. In recent years, unisexual cultivation has developed rapidly due to the development of sex-specific markers [20]. Sex determination genes and

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad differentiation pathways can help researchers screen the sex-specific markers in some aquaculture fishes [21]. For instance, sex-specific markers were found from dmrt1 gene of the yellow drum (Nibea albiflora), amhr2 gene of Patagonian pejerrey (Odontesthes hatcheri) and amhr2 gene of pufferfish (Takifugu obscurus) [22][23][24]. Conventional technologies, such as AFLP and RAPD, have already screened sex-specific markers in some aquatic species [25,26]. However, conventional technologies are time-consuming, and cannot detect all the sex-specific markers in fishes [26]. Consequently, high-throughput methods of NGS technology are the best choice to verify sex-specific markers and sequences. The sex-specific markers of American shad are still unclear. In the research, we created an efficient testing process to identify male-specific tags and male-specific SNPs. Male-specific tags were identified in 60 samples of two generations of fish. Sex-specific markers can help researchers identify the sex-related genes in American shad. American shad is an essential economic fish species and widely farmed in China. However, the culture model of all female fish increases the production efficiency of American shad. The verification of sex-specific markers can help promote the process of genetic breeding [27]. The technology of mono-sex breeding for the mass production of economic fish is developing rapidly [28]. In this context, sex-specific molecular markers of American shad are urgently needed. All-female production technology has been developed and successfully applied in yellow catfish [29][30][31]. However, sex determination is a complex process, which has its unique evolutionary patterns and it also can be affected by external factors, including genetic, environmental (e.g., temperature), behavioral, and physiological factors [32]. For example, Baroiller et al. (2009) found that the

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad sex determination system of the Nile tilapia (Oreochromis niloticus), both domestic and wild populations, was affected by the interactions between genetic determination and temperature [33]. We can achieve sex reversal through hormone feeding and breed XX neo-males, producing an all-XX female brood. In conclusion, five male-specific tags are identified, and the XX/ XY sex system of American shad is predicted. The results of PCR and agarose gel electrophoresis were validated in two generations. However, the sex-specific markers of American shad are still unclear. The sex determination and differentiation pathway genes associated with the five male-specific tags should be identified. Those genes combined with sex-specific markers are important for developing sex-controlled breeding to generate all-female populations in the aquaculture industry.

Conclusions
This is the first study to identify sex-specific markers using 2b-RAD sequencing in American shad. The preliminary screening of eleven male-specific tags ( Table 2) and three male heterogametic SNP loci were screened in twenty fish using 2b-RAD sequencing. The male-specific sequences were used to design sex-specific primer pairs, and five tags were finally identified by PCR in the second generation. The results indicate that five male-specific tags can accurately identify sex in American shad. The research created an efficient technological means for identifying the genetic sex of American shad and for the quantity production of all-female fish.

PLOS ONE
Sex-specific sequences through 2b-RAD sequencing in American shad Supporting information S1 Table. Primers used to verify candidate male-specific 2b-RAD-tags and sex-specific SNP locus. (DOCX) S1 Text. The details of library preparation and data analysis.